\
PERGAMON International Journal of Immunopharmacology 10 "0888# 16Ð35
Melatonin administration in tumor!bearing mice "intact and pinealectomized# in relation to stress\ zinc\ thymulin and IL!1 E[ Mocchegiania\ \ L[ Perissinb\ L[ Santarellia\ A[ Tibaldia\ S[ Zorzetc\ V[ Rapozzib\ R[ Giacconia\ D[ Buliana\ T[ Giraldic a Immunolo`y Center\ Gerontolo`y Research Department\ Italian National Research Centres on A`in`\"I[N[R[C[A[#\ Ancona\ Italy b Department of Biomedical Sciences and Technolo`ies\ University of Udine\ Udine\ Italy c Department of Biomedical Sciences\ University of Trieste\ Trieste\ Italy
Received 10 May 0887^ accepted 09 August 0887
Abstract
Melatonin "MEL# may counteract tumors through a direct oncostatic role[ MEL is also an antistress agent with immunoenhancing properties against tumors due to a suppressive role of MEL on corticosterone release[ Rotational stress "RS# "spatial disorientation# facilitates metastasis progression in mice[ Also\ MEL counteracts tumors because of its in~uence on immune responses via the metabolic zinc pool\ which\ is reduced in tumors and stress[ Zinc is required for normal thymic endocrine activity "i[e[ thymulin# and interleukin!1 "IL!1# production[ Because in vivo data is still controversial\ exogenous MEL treatment "11 days in drinking water# in both intact and pinealectomized "px# mice bearing Lewis lung carcinoma leads to signi_cant decrements of metastasis volume\ restoration of the negative crude zinc balance\ recovery of thymulin activity and increment of IL!1 exclusively in intact and px tumor bearing mice subjected to RS[ Signi_cant inverse correlations are found in both stressed intact and px tumor bearing mice after MEL treatment between zinc and corticosterone "r 9[67\ P ³ 9[90^ r 9[79\ P ³ 9[90\ respectively#[ Positive correlations between zinc and IL!1 "r 9[64\ P ³ 9[90^ r 9[62\ P ³ 9[90\ respectively# or thymulin "r 9[64\ P ³ 9[90^ r 9[71\ P ³ 9[90\ respectively# are observed in same models of mice[ These _ndings suggest a MEL action to decrease metastasis mediated by a possible interplay between zinc and MEL\ via corticosterone\ with consequent restoration of thymic e.ciency and IL!1 production[ MEL as an antistress agent with immunoenhancing properties in cancer deserves further consideration[ Þ 0888 International Society for Immunopharmacology[ Published by Elsevier Science Ltd[
Key words] Zinc^ Melatonin^ Stress^ Metastasis^ Thymulin^ Corticosterone^ IL!1
Abbreviations] AAS\ atomic absorption spectrophotometry^ ELISA\ enzyme!linked immunoabsorbent assay^ FTS\ inactive zinc!unbound thymulin^ I\ intact mice^ IL!1\ interleukin!1^ MEL\ melatonin^ MP\ metabolic period^ NF!kB\
Corresponding author[ Tel] ¦28 60 7993005^ Fax ¦28 60 195680^ E!mail] e[mocchegianiÝinrca[it
9081Ð9450:88:,08[99 Þ 0888 International Society for Immunopharmacology[ Published by Elsevier Science Ltd[ PII] S 9 0 8 1 Ð 9 4 5 0 " 8 7 # 9 9 9 5 6 Ð 7 17 E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35 nuclear factor!kb^ POMC\ proopiomelanocortin^ Px\ pinealectomized mice^ RIA\ radioimmunoassay^ RS\ rotational stress^ SDI\ stressed intact mice^ SDPx\ stressed pinealectomized mice^ TNF!a\ tumor necrosis factor!a^ ZnFTS\ active zinc!bound thymulin^ ZnFTS¦FTS\ total thymulin[
0[ Introduction
Melatonin "MEL# counteracts tumors with a direct oncostatic role or as an antistress agent with immunoenhancing properties[ In vitro MEL inhibits tumor growth "Shellard\ Whelan + Hill\ 0878^ Cos + Blask\ 0883^ Cos\ Fernandez + Sanchez!Barcelo 0885#[ Leukemia bearing mice have shown an antistress role of MEL with immunoenhancing properties against tumors because of a suppressive action of MEL on corticosterone "Maestroni + Conti\ 0889#[ Rotational Stress "RS# facilitates the progression of tumors in mice\ via neuroendocrine!immune modulation e}ectors of the host "Justice\ 0874^ Vogel + Bower\ 0880^ Giraldi\ Perissin\ Zorzet\ Piccini + Rapozzi\ 0878#[ In turn\ RS a}ects MEL production in BD1F0 mice "Giraldi\ Perissin\ Zorzet + Rapozzi 0883a^ Giraldi\ Perissin\ Zorzet\ Rapozzi + Rodani\ 0883b^ Perissin\ Zorzet\ Rapozzi\ Paoletti + Giraldi\ 0883#\ enhancing circulating MEL in hybrid BD1F0 mice "Perissin\ Zorzet\ Rapozzi + Giraldi\ 0882#\ despite no MEL induction has been reported in C46BL:5 mice "Ebihara\ Marks\ Hudson + Menaker\ 0875#[ MEL action against tumors also occurs because of MEL|s immunomodulatory role "Caroleo\ Frasca\ Nistico| + Doria\ 0881^ Maestroni\ 0882^ Pierpaoli + Regelson\ 0883^ Conti + Maestroni 0884#[ In vivo and in vitro data have recently shown MEL to a}ect thymic endocrine activity "i[e[ thymulin# and interleukin!1 "IL!1#\ in young pinealectomized "px# and old mice by means of metabolic zinc pool turnover\ via glucocorticoids pathway[ The interplay between zinc and MEL\ via corticosterone\ for normal immune e.ciency has assigned to MEL a role as an immunomodulating agent "Mocchegiani et al[\ 0883a\ 0885\ 0887#[ Zinc de_ciency\ impaired thymulin activity and altered serum IL!1 occur both in cancer and stress "Fabris + Mocchegiani\ 0884#[ Zinc is required both for thymulin activity which regulates T!cell mediated immunity "Dardenne et al[\ 0871# and for IL!1 production by lymphocytes "Tanaka\ Shiozawa\ Moromoto + Fujita\ 0889#[ Circulating IL!1 is also a good marker to test immune impairment in zinc!de_cient cancer patients "Prasad\ Beck\ Grabowski\ Kaplan + Plathog\ 0886a#[ With these premises\ MEL administration was carried out in px and intact mice bearing Lewis lung carcinoma subjected or not to RS\ and metastasis volume\ metabolic zinc pool\ thymulin activity\ corticosterone and IL!1 plasma levels were measured in order to further verify MEL|s antistress role with immunoenhancing properties "Maestroni + Conti\ 0889# and:or its direct oncostatic role "Shellard et al[\ 0878^ Cos + Blask\ 0883^ Cos et al[\ 0885#[ MEL action against tumors is still controversial and unde_ned "Stanberry\ Das Gupta\ + Beattie\ 0872^ Blask\ 0873^ Slominski + Pruski\ 0882^ Giraldi et al[\ 0883a\b^ Karasek\ 0886# and requires urgent clari_cation[
1[ Experimental procedures
1[0[ Animals\ tumor transplantation and measurements
Female BD1F0 mice "C46BL:5×DBA:1 F0# "1 months of age# "Charles River\ Calco\ Como\ Italy# weighing 07Ð10 g\ were used[ Lewis lung carcinoma was provided by the National Cancer E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35 18
Institute\ Bethesda MD\ U[S[A[ The subcutaneous propagation of tumor in BD1F0 mice\ as well as the measure of primary tumor volume and lung metastasis were performed as described elsewhere "Giraldi et al[\ 0878^ Giraldi et al[\ 0883a\b#[ All animals were weighed before the experiment and at sacri_ce to determine whether the e}ects seen were related to dietary intake "Mocchegiani et al[\ 0887#[ Hybrid BD1F0 mice were chosen because the syngenic implantation of Lewis lung carci! noma\ whose malignant progression is sensitive to RS\ works well in this strain "Giraldi et al[\ 0878#[
1[1[ Animal housing and rotational stress Mice\ with drinking water and pellet food "Nossan\ Italy# ad libitum\ were kept 4 per cage in order to avoid the e}ects of overcrowding or isolation on tumor progression "Riley\ Fitzmaurice + Spackman\ 0870#[ The cages were placed in a low housing stress "protected# environment for 1 weeks before tumor inoculation\ in order to allow the animals to recover from the stress of shipment "Riley et al[\ 0870#[ As such\ mice become adapted to the new housing conditions[ Temperature and humidity were constant "19>C and 59)\ respectively#[ The cycle of illumination was 01:01 h[ "lights on from 9799Ð1999 h# with an intensity of 1999 lux in the cages[ RS "spatial disorientation# was applied to the animals in the low stress environment by spinning the cages at 34 rpm for 09 min every hour from time of tumor inoculation until sacri_ce\ as described elsewhere "Giraldi et al[\ 0878^ 0883b#[ RS is a good model for stress and tumor progression studies in animals "Riley et al[\ 0870^ Justice\ 0874^ Giraldi et al[\ 0878^ 0883a\b#[
1[2[ Pinealectomy and melatonin administration Surgical pinealectomy was performed using a procedure described previously "Moechegiani et al[\ 0885#[ A post mortem inspection of the brain was performed at sacri_ce in order to exclude in px animals pineal gland presence[ The pinealectomy was performed on mice at 1 months of age[ Tumour inoculation and melatonin treatment were performed 1 months later because 1 months is su.cient time to allow for the disappearance of endogenous nocturnal peak of MEL in inbred mice "Conti + Maestroni 0885#[ MEL "0[14 mg:Kg:night# was administered into the drinking water for 11 days in order to see MEL e}ects on metastasis volume in relation to RS in tumor bearing BD1F0 mice "Giraldi et al[\ 0883a\b#[ MEL "5[1 mg:l# was dissolved in water containing 9[1) ethanol[ The MEL!water was administered during the dark:night period "1999Ð97]99 h#\ from tumor inoculation until sacri_ce[ MEL!containing bottles were removed from 9799Ð1999 h[ Normal tap water was administered during this light:day period[ MEL treatment\ tumor inoculation and rotational stress were carried out in mice at the age of 3 months[ Primary tumor volume was determined on day 03 and lung metastasis were measured at sacri_ce on day 11\ respectively\ from tumor inoculation[ The age of mice "experimental groups and controls# averaged 3[6 months at sacri_ce[ The experimental model was carried out in MayÐJune because of seasonal variations of MEL and the e}ect of RS on metastasis volume were more evident in spring and summer "Perissin et al[\ 0883#
1[3[ Metabolic zinc assessment The metabolic period "MP# for zinc assessment was performed for 6 days before the sacri_ce "Mocchegiani et al[\ 0883a#[ The measurements of food and water intake and of urinary and faecal 29 E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35 excretion were performed every day for each mouse[ Zinc was measured in food and water\ and the zinc intake was calculated on the basis of food and water consumption per day[ Zinc was measured in urine and faeces and the zinc excretion was calculated on the basis of urine and faeces excretion per day[ The di}erence between zinc intake and zinc excretion is the crude zinc balance "metabolic zinc pool# "Mocchegiani et al[\ 0883a#[ In order to avoid the stress of placing the mice in metabolic cages\ urine and faeces over 13 h were collected on Wathman 2MM chromatographic paper placed below a stainless steel grid and covering the bottom of the cages "Perissin et al[\ 0882#[
1[4[ Zinc determination
1[4[0[ Blood Heparinized blood samples for zinc measurements were collected in ~uorinated tubes "No[ 004206\ LP\ Italy# and centrifuged 19 min later at 2999 g for 09 min[ Zinc was determined by A[A[S[ according to the method and zinc references standard of Fernandez + Khan "0861#[
1[4[1[ Urine Zinc determination in urine samples was carried out over 13 h by A[A[S[ using a dry!washing technique and 4 ml of HCl 5N for zinc extraction\ according to the method and zinc references standard\ as suggested by Chapman + Pratt "0850#[
1[4[2[ Faeces The determination of zinc in faeces was carried out by A[A[S[ according to the method of Lodefoged
"0879#[ Brie~y\ 1 g of faeces was boiled for 29 min in 04 ml of a mixture of HCl!HNO2 and deionized water in the proportion 16]2]19[ After boiling\ the mixture was diluted to 49 ml with deionized water\ and then a reading was taken at A[A[S[ using zinc references standard "Lodefoged\ 0879#[
1[4[3[ Food The method of Lodefoged "0879# was used to analyse zinc in food[ Brie~y\ 14 ml of hydrochloric acid solution was added to 4 g of food sample\ brought to boiling point and _ltered through No[ 0 Whatman _lter paper[ The _ltrate was directly analysed against the zinc references standard "Lodefoged\ 0879#[
1[4[4[ Water The determination of zinc in the drinking "tap# water was performed by A[A[S[ using the method and zinc references standard of Fishman + Downs "0855#[
1[5[ Thymulin determination
This technique\ originally developed by Bach\ Dardenne\ Pleau and Bach "0864# and described elsewhere "Mocchegiani et al[\ 0883a\b#\ is speci_c for zinc!bound active thymulin "ZnFTS#\ because the assay is una}ected by other thymic hormones\ and the rosette!inducing activity is completely removed by passing plasma samples through an antithymulin immunoadsorbent[ The sensitivity of the bioassay makes it possible to detect 0 pg:ml of synthetic thymulin "Sigma\ U[S[A[#[ E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35 20
The assay is reliable^ in two consecutive blind assays a di}erence no greater than 0:log1 was found in all samples[ In order to avoid possible interference by zinc bioavailability\ thymulin measurements were performed concomitantly with the in vitro addition of zinc sulphate to the plasma samples at a _nal concentration of 199 nM\ which is the optimal concentration for unmasking zinc!unbound inactive thymulin "FTS#[ As such\ the total amount of thymulin produced "zinc!bound ZnFTS¦zinc!unbound FTS# can be evaluated in the circulation "Mocchegiani et al[\
0883a\b#[ Thymulin activity is expressed as 0:log1[ The apparently low molar concentration of zinc required may be explained by the fact that the bioavailable free zinc is no more than 1Ð2) of total plasma zinc\ the majority being bound to proteins which are retained by the 49\999 mol wt cut! o} membranes "Mocchegiani et al[\ 0883a\b#[ This bioassay is still required because thymulin radioimmunoassay developed recently is unable to discriminate between zinc!bound and zinc! unbound thymulin "Sa_eh et al[\ 0889#[
1[6[ Corticosterone determination
Plasma corticosterone was tested in blood samples collected between 9899 and 0999 h by intracardiac puncture immediately after sacri_ce by cervical dislocation\ with the greatest care being taken not to cause further stress by these procedures[ Moreover\ mice were sacri_cied in a separate room from others in the sequence] control mice\ tumor!bearing mice\ and tumor!bearing mice¦MEL\ in order to avoid the possibility of stress caused by the sacri_ce "Mocchegiani et al[\ 0887#[ The analysis was determined by using RIA rat!corticosterone!2H kit "ICN Biomedicals\ Inc\ CA\ U[S[A[# which used a speci_c anti!corticosterone antibody[ The data obtained "expressed in ng:ml# were referred against a standard curve[ The percentage of cross reaction with other steroids was ³9[90[ The sensitivity was of 9[94 ng:ml of corticosterone[
1[7[ Interleukin!1 determination
Interleukin 1 "IL!1# was measured in plasma samples by means of an enzyme!linked immu! noabsorbent assay "Inter Test 1X Mouse IL!1 ELISA kit\ Genzyme\ U[S[A[# based on a sandwich principle using monoclonal anti!mouse"m#IL!1 in conjunction with biotinylated polyclonal anti! mIL!1 antibody[ A peroxidase!conjugate\ streptavidin\ which binds to biotin!tagged immune complexes captured in the plate\ was used[ The results\ expressed in pg:ml\ correspond to the average of two separate assays[ The speci_city of the results was con_rmed using an Inter Test!1X kit with other mouse or rat recombinant cytokines that did not produce absorbances above the 9 pg:ml standard[ A standard curve was obtained by plotting the concentrations of mIL!1 standards vs their resulting absorbances[ The mIL!1 concentrations in experimental samples were determined from the standard curve[ The sensitivity of the ELISA mIL!1 kit ranged between 04 and 859 pg:ml[
1[8[ Statistical analysis
The signi_cance of the di}erences between means was assessed using paired Student|s t!tests\ MannÐWhitney tests and ANOVA tests "one!way#[ Correlations were determined via linear regression analysis by the least!square method[ The di}erence between the various regression lines was evaluated by analysis of covariance[ Di}erences were considered signi_cant when P ³ 9[94[ 21 E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35
2[ Results
2[0[ Pinealectomy\ melatonin\ tumor progression and rotational stress
Pinealectomy\ melatonin treatment or RS do not cause any signi_cant e}ect on primary tumor growth in mice bearing Lewis lung carcinoma "Table 0#[ In contrast\ RS increases the volume of lung metastasis in intact mice as compared to respective controls "035[7 vs 58[5# "P ³ 9[94# "Table 0#[ Surgical pinealectomy does not modify metastasis volume in non!stressed mice "015[5 vs 58[5#[ Signi_cant increments of metastasis volume are observed in px stressed mice\ as compared to respective controls "275[9 vs 015[5# or to intact stressed controls "275[9 vs 035[7# "P ³ 9[94# "Table 0#[ MEL treatment is ine}ective in reducing metastasis volume in non!stressed mice\ with or without pinealectomy "064[5 vs 58[5^ 111[3 vs 015[5\ respectively# "Table 0#[ MEL treatment is e}ective in reducing metastasis volume in intact stressed "003[7 vs 035[7# and in px stressed mice "005 vs 275# "P ³ 9[94# as compared to respective controls "Table 0#[ In~uences of dietary intake as well as of RS on body weight are avoided because of no body weight di}erences between treated and untreated MEL tumor bearing mice "intact and px mice with or without RS# at sacri_ce as compared to values observed at the beginning of experiment "data not shown#[ The night!MEL drinking water uptake is 3[7Ð4 ml:night:mouse[ No di}erences are found for this parameter between stressed and non!stressed mice "intact and px# and respective controls[ No signi_cant
Table 0 E}ect of pinealectomy\ exogenous melatonin administration and rotational stress in primary tumor growth and lung metastasis formation
Mice Mel RS Primary tumor Metastasis volume volume "cm2# "mm2#
Intact − − 2[429[31 58[5204[1 Px − − 2[529[26 015[5220[3 Intact ¦ − 1[829[05 064[5235[2 Px ¦ − 1[829[15 111[3238[4 Intact − ¦ 2[429[25 035[7213[4 Px − ¦ 1[829[13 275[9202[1 Intact ¦ ¦ 2[029[18 003[7205[5 Px ¦ ¦ 2[029[12 005[9206[9 ShamÐpx − − 2[429[10 63[128[1¦ ShamÐpx − ¦ 2[129[08 051[5204[1
09 animals for each experimental group] 4 animals for cage[ RS Rotational stress Mean2SD[ P ³ 9[94 when compared to the values of non!stressed controls "035[7 vs 58[5# "MannÐWhitney test#[ P ³ 9[94 when compared to values of Px alone "275[9 vs 015[5# or to RS alone "275[9 vs 035[7# "MannÐWhitney test#[ P ³ 9[94 when compared to Px stressed mice "005 vs 275[9# or to intact stressed mice "003[7 or 094[9 vs 035[7# "MannÐWhitney test#[ No signi_cant di}erences exist in primary tumor growth and in metastasis volume between no stressed\ shamÐpx and intact mice "63[1 vs 58[5# and between stressed shamÐpx and intact mice "051[5 vs 035[7# after two months from pinealectomy\ whereas signi_cant di}erences exist between non!stressed and stressed shamÐpx mice "¦P ³ 9[90# "63[1 vs 051[5# "MannÐWhitney test#[ E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35 22
Table 1 Pinealectomy\ melatonin treatment and neoplasia] zinc metabolism
Crude zinc balance Zincaemia "mg:day:mouse# "mg:dl#
A Intact ¦0[529[4 069[121[7 Intact¦tumor −1[129[7 59[028[3 Intact¦tumor¦MEL −0[9429[6 45[628[6
B ShamÐpx ¦0[229[5 047[322[6 ShamÐpx¦tumor −1[929[8 52[326[6 Px −0[829[7 77[624[9 Px¦tumor −3[920[9 56[4200[9 Px¦tumor¦MEL −2[929[7 45[128[6
09 animals for each experimental group[ Mean¦SD[ P ³ 9[990 when compared to intact\ Px control\ shamÐpx\ respectively[
di}erences in volume metastasis are observed between intact and shamÐpx tumor bearing mice subjected or not subjected to RS "Table 0#[ Signi_cant di}erences are observed in shamÐpx tumor bearing mice subjected to RS as compared to non RS subjected mice "051[5 vs 63[1# "P ³ 9[90#\ as it occurs in intact stressed mice "035[7 vs 58[5# "P ³ 9[94# "Table 00#[
2[1[ Pinealectomy\ MEL and crude zinc balance
Crude zinc balance is positive in intact mice "Table 1A#[ The tumor induces a negative crude zinc balance with reduced zincaemia in intact mice as compared to respective controls[ The crude zinc balance is negative both in px controls and in px tumor bearing mice "Table 1B#[ MEL treatment is ine}ective in restoring a crude zinc balance and plasma zinc levels in both tumor bearing intact and px mice "Table 1A\B#[ More marked negative crude zinc balance and more reduced zincaemia are observed in presence of tumor "px and intact# "Table 1A\B#[ The crude zinc balance is negative despite the zinc content in Nossan "Como\ Italy# food being 197 pp:m[ No di}erences exist between intact and shamÐpx mice in presence:absence of tumor\ respectively "Table 1A\B#[
2[2[ Pinealectomy\ MEL\ crude zinc balance and rotational stress
The crude zinc balance is negative in stressed intact controls whereas plasma zinc levels are in the normal range "Table 2A#[ The tumor induces negative crude zinc balance associated with low plasma zinc levels "Table 2A#[ MEL treatment restores crude zinc balance as well as plasma zinc levels "Table 2A#[ Px stressed mice show a negative crude zinc balance which is more marked in 23 E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35
Table 2 Pinealectomy\ melatonin treatment\ neoplasia and rotational stress] zinc metabolism
Crude zinc balance Zincaemia "mg:day:mouse# "mg:dl#
A Stressed intact controls −0[829[6 029[9209[9 Stressed intact¦tumor −1[729[7 47[323[5 Stressed intact¦tumor¦MEL ¦9[129[6 84[923[2
B Stressed ShamÐpx −0[729[6 019[6201[9 Stressed ShamÐpx¦tumor −1[429[5 52[925[6 Stressed Px −1[329[7 74[923[7 Stressed Px¦tumor −3[629[8 57[924[5 Stressed Px¦tumor¦MEL ¦1[929[7 098[724[8
09 animals for each experimental group[ Mean2SD[ P ³ 9[990 when compared to stressed intact\ stressed Px control\ shamÐpx\ respectively[
the presence of tumor "Table 2B#[ MEL treatment restores crude zinc balance as well as plasma zinc levels in stressed px tumor bearing mice "Table 2B#[ No di}erences exist in negative crude zinc balance between stressed shamÐpx and intact mice in presence:absence of tumor\ respectively "Table 2A\B#[
2[3[ Pinealectomy\ MEL\ and thymic endocrine activity
Active thymulin "ZnFTS# and total thymulin "active ZnFTS¦inactive FTS# plasma levels are in the normal range for the age in intact "I# mice "Fig[ 0A#[ The tumor leads to a signi_cant reduction of ZnFTS as compared to intact controls "P ³ 9[990#\ whereas total thymulin levels are not a}ected by the tumor "Fig[ 0A#[ MEL treatment is ine}ective in restoring ZnFTS in tumor bearing mice "Fig[ 0A#[ Plasma ZnFTS levels are low in px mice as compared to intact controls "P ³ 9[990# "Fig[ 0B#[ Tumour and MEL treatment do not a}ect reduced ZnFTS levels in px mice "Fig[ IB#[ Total thymulin levels are in the normal range for the age in px mice with no modi_cations by tumor or MEL treatment "Fig[ 0B#[ No signi_cant di}erences exist in active and total thymulin between intact and shamÐpx mice in presence:absence of tumor\ respectively "Fig[ 0A\B#
2[4[ Pinealectomy\ MEL\ thymic endocrine activity and rotational stress
Stressed intact mice without tumor "SDI# show no signi_cant modi_cations of ZnFTS and total thymulin plasma levels as compared to intact controls "I# "Fig[ 1A#[ The tumor causes a marked reduction of ZnFTS "P ³ 9[990#\ whereas total thymulin is not modi_ed "Fig[ 1A#[ MEL treatment restores ZnFTS "Fig[ 1A#[ Px stressed mice "SDPx# show decreased ZnFTS as compared to SDI E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35 24
Fig[ 0[ Plasma active thymulin "ZnFTS# "Ž# and total thymulin "ZnFTS¦FTS# "Á# in intact "I# mice "Fig[ 0A# and in pinealectomized "Px# mice "Fig[ 0B# after injections of Lewis lung carcinoma and the e}ect of melatonin treatment in both models of tumor bearing mice[ Thymulin activity is expressed in 0:log1[ P³9[990 when compared to active thymulin of intact or shamÐpx controls "I# Mean¦SD[
mice "P ³ 9[94#[ More marked reduction of ZnFTS is induced by the tumor in SDPx mice as compared to SDPx controls "P ³ 9[94# "Fig[ 1B#[ MEL treatment increases ZnFTS in SDPx tumor bearing mice "Fig 1B#[ No di}erences in total thymulin are observed in these three px mice groups 25 E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35
Fig[ 1[ Plasma active thymulin "ZnFTS# "Ž# and total thymulin "ZnFTS¦FTS# "Á# in stressed intact mice "SDI# "Fig[ 1A# and in stressed pinealectomized mice "SDPX# "Fig[ 1B# after injections of Lewis lung carcinoma and the e}ect of melatonin treatment in both models of stressed tumor bearing mice[ Thymulin activity is expressed in 0:log1[ P³9[990 when compared to active thymulin of SDI or SD shamÐpx controls^ P ³ 9[94 when compared to active thymulin of SDI or SD shamÐpx controls^ P ³ 9[94 when compared to active thymulin of SDPx controls[ Mean¦SD[ E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35 26
"Fig[ 1B#[ Signi_cant positive correlations are found between ZnFTS and zinc in both stressed tumor bearing intact and px mice treated with MEL "r 9[64\ P ³ 9[90^ r 9[71\ P ³ 9[90\ respectively#\ whereas they are absent in non!stressed tumor bearing mice treated with MEL "intact and px#[ No signi_cant di}erences exist in active and total thymulin between SDI and stressed "SD# shamÐpx mice in presence:absence of tumor\ respectively "Fig[ 1A\B#[
2[5[ Corticosterone
Corticosterone is increased in px mice as compared to intact mice "129 vs 024# "P ³ 9[990# "Table 3A\B#[ Tumour and MEL treatment do not lead to any signi_cant modi_cations of corticosterone in intact and px mice as compared to respective controls "Table 3A\B#[ RS induces signi_cant increments of corticosterone in intact mice as compared to intact controls "066 vs 024# "P ³ 9[94#\ whereas the increased levels of corticosterone in px mice are not a}ected by RS "139 vs 129# "Tables 3 and 4A\B#[ The tumor does not a}ect corticosterone in stressed intact mice "062[8 vs 066#\ whereas it leads to signi_cant increments exclusively in px stressed mice as compared to px stressed controls "229[6 vs 139# "P ³ 9[90# "Table 4A\B#[ MEL treatment reduces corticosterone in both stressed tumor bearing intact and px mice "098[3 vs 062\8^ and 166 vs 229[6\ respectively# "P ³ 9[90# "Table 4A\B#[ A trend to corticosterone reduction is also observed after MEL treatment in both non!stressed tumor bearing intact and px mice "017 vs 025 and 177[3 vs 200[5\ respectively# "Table 3A\B#[ Signi_cant inverse correlations are found between corticosterone and zinc in both stressed tumor bearing intact and px mice treated with MEL "r −9[67\ P ³ 9[90^ r −9[79\ P ³ 9[90\ respectively#\ whereas they are absent in non!stressed tumor bearing mice treated with MEL "intact and px#[ The stress interference due to pinealectomy is avoided because of no signi_cant corticosterone di}erences between shamÐpx and intact mice\ and between stressed shamÐpx and
Table 3 Pinealectomy\ melatonin treatment and neoplasia] corticosterone plasma levels
Corticosterone "ng:ml#
A Intact 02422[4 Intact¦tumor 02523[3 Intact¦tumor¦MEL 01722[4
B ShamÐpx 01722[7 ShamÐpx¦tumor 027200[8 Px 129215 Px¦tumor 200[5214[0 Px¦tumor¦MEL 177[3236[1
P ³ 9[990 when compared to intact control and shamÐpx mice[ 27 E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35
Table 4 Pinealectomy\ melatonin treatment\ neoplasia and rotational stress] cort! icosterone plasma levels
Corticosterone "ng:ml#
A Stressed intact 066[9217 Stressed intact¦tumor 062[8216 Stressed intact¦tumor¦MEL 098[3207[1
B Stressed ShamÐpx 070[9218 Stressed ShamÐpx¦tumor 071[0220 Stressed Px 139[9215 Stressed Px¦tumor 229[6213[1 Stressed Px¦tumor¦MEL 166[1210
P ³ 9[94 when compared to non!stressed intact control "Table 4A#[ P ³ 9[90 when compared to Px controls and shamÐpx¦tumor[ P ³ 9[90 when compared to stressed intact\ stressed shamÐpx or stressed Px controls\ respectively[
intact mice in presence:absence of tumor\ respectively\ as well as between px and px stressed mice "Table 3 and 4#[
2[6[ Interleukin!1 "IL!1#
IL!1 plasma levels are reduced in both intact and stressed tumor bearing mice as compared to respective controls "P ³ 9[990# "Fig[ 2A\ panels 0 and 1# with no modi_cations after MEL treatment "Fig[ 2A\ panel 0#[ Signi_cant increments are observed in stressed tumor bearing intact mice "SDI¦tum[# after MEL treatment "P ³ 9[94# "Fig[ 2A\ panel 1#[ The same recovery is also achieved in stressed tumor px mice "SDPx¦tum¦MEL# as compared to respective controls "SDPx or SDPx¦tum# "P ³ 9[94# "Fig[ 2B\ panel 3#[ Tumour and MEL treatment do not a}ect reduced IL! 1 plasma levels present in px mice "Fig[ 2B\ panel 2#[ Signi_cant positive correlations are found between zinc and IL!1 in both stressed tumor bearing intact and px mice treated with MEL "r 9[64\ P ³ 9[90^ r 9[62\ P ³ 9[90\ respectively#[ Signi_cant inverse correlations are found between corticosterone and IL!1 in both stressed tumor bearing intact and px mice treated with MEL "r −9[69^ P ³ 9[94^ r −9[57^ P ³ 9[94\ respectively#\ whereas they are absent in non! stressed tumor bearing mice treated with MEL "intact and px#[ No signi_cant di}erences exist in IL!1 between intact and shamÐpx mice "Fig[ 2A\B\ panels 0 and 2# as well as between SDI and SD shamÐpx mice "Fig[ 2A\B\ panels 1 and 3# in presence:absence of tumor\ respectively[ E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35 28
Fig[ 2[ A# Plasma IL!1 levels in intact mice "I# "panel 0# and in stressed intact mice "SDI# "panel 1# after injections of Lewis lung carcinoma and the e}ect of melatonin treatment in both intact tumor bearing mice "no stressed and stressed mice# "panels 0 and 1#[ B# Plasma IL!1 levels in pinealectomized "Px# "panel 2# and in stressed pinealectomized mice "SDPx# "panel 3# after injections of Lewis lung carcinoma and the e}ect of melatonin treatment in both px tumor bearing mice "no stressed and stressed px mice# "panel 2 and 3#[ P ³ 9[990 when compared to intact controls "I#^ P ³ 9[94 when compared to SDI or SDPx control mice\ respectively[ Mean¦S[D[ 39 E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35
3[ Discussion
RS leads to signi_cant increments of metastasis volume associated with impaired ZnFTS and decreased IL!1 production which is more evident in px stressed tumor bearing mice[ MEL decreases metastasis volume and restores ZnFTS and IL!1 exclusively in stressed intact and px tumor bearing mice[ The use of MEL as an anti!stress agent with immunoenhancing properties against tumors deserves more consideration[ RS facilitates tumor progression in mice\ via neuroendocrine!immune modulation e}ectors of the host "Justice\ 0874^ Giraidi et al[\ 0878#[ MEL as an antistress agent with immunoenhancing properties\ particularly on Th0¦ cells\ via opiatergic mechanism\ has been documented in leukemia bearing mice "Maestroni + Conti\ 0889#\ because of suppressive role of MEL on corticosterone release "Gupta\ 0889^ Arendt\ 0883#[ In vitro models show MEL|s direct oncostatic role because it inhibits DNA synthesis in cancer cell lines "Hill + Blask\ 0877^ Shellard et al[\ 0878^ Helton + Kane\ 0880^ Stankov et al[\ 0880^ Pickering + Niles\ 0881^ Cos + Blask\ 0883^ Cos et al[\ 0885# In vivo experiments are\ by contrast\ controversial and unde_ned "Stanberry et al[\ 0872^ Blask\ 0873^ Slominski + Pruski\ 0882^ Giraldi et al[\ 0883a#[ MEL|s oncostatic action may be due to its immunomodulatory role "Caroleo et al[\ 0881^ Maestroni\ 0882^ Pierpaoli + Regelson\ 0883^ Skwarlosonta\ 0885#[ Direct and indirect mechanisms acting on the immune system have been proposed[ MEL receptors on lymphocytes "Lopez! Gonzales\ Calvo\ Osuna + Guerrero\ 0881#\ in the thymus "Martin!Cacao et al[\ 0882^ Pozo\ Delgado\ Calvo\ Garcia!Perganeda + Guerrero\ 0886# and at the nuclear level "Calberg\ Wein! semberg + Schrader\ 0886# have been found[ Indirect mechanisms involving the neuroendocrine! immune interactions "Maestroni 0882^ Gupta\ 0889# or the proopiomelanocortin "POMC# gene expression "Gupta\ Ghose + Revskoy\ 0883#\ may be added[ Among the latter\ the metabolic zinc pool has also been proposed "Mocchegiani et al[\ 0883a\ 0885\ 0887#\ because of the relevance of zinc for normal immune response "Chandra\ 0874#\ including thymic endocrine activity[ Indeed zinc is required to confer biological activity to thymulin "ZnFTS#\ which in turn regulates T!cell mediated immunity "Dardenne et al[\ 0871#[ The zinc!unbound form "FTS# is inactive with an inhibitory action on the active form "ZnFTS# "Mocchegiani et al[\ 0883b#[ The in vitro addition of zinc to plasma samples containing FTS unmasks the inactive form showing the total amount "active ZnFTS¦inactive FTS# of thymulin molecules produced "Mocchegiani et al[\ 0883b#[ This occurs in marginal zinc de_ciencies\ including cancer "Moechegiani et al[\ 0883b#[ Supplementing zinc restores central and peripheral immune de_cits\ suggesting that the thymic failure is not intrinsic but largely dependent on low peripheral zinc bioavailability to saturate all thymulin molecules produced "Fabris + Mocchegiani\ 0884#[ Zinc is also crucial for the glucocorticoids pathway "Prasad\ 0874#[ Because of MEL|s suppressive e}ect on corticosterone "Maestroni + Conti 0889\ Gupta\ 0889\ Arendt\ 0883#\ and the action of MEL on the metabolic zinc pool is mediated by glucocorticoids\ an interplay between zinc and MEL\ via glucocorticoids\ has been proposed during development and aging for normal immune e.ciency with the role of MEL as an immunomodulating agent "Mocchegiani et al[\ 0883a\ 0885\ 0887#[ Lewis lung carcinoma leads to negative crude zinc balance in both models of mice "intact and px#\ despite the zinc content in Nossan food "197 ppm#\ perhaps because of an intestinal malabsorption in cancer "Duncan + Dreosti\ 0864#[ ZnFTS levels are reduced with no modi_cations after MEL treatment[ Total thymulin level is not a}ected by the tumor or by MEL treatment[ E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35 30
Thus\ the thymulin defect is triggered because of low zinc peripheral bioavailabilty to saturate all thymulin molecules produced in tumor bearing mice\ as in human cancer "Mocchegiani et al[\ 0883b#[ In contrast\ MEL treatment restores crude zinc balance\ ZnFTS and corticosterone in stressed intact and px mice independent of the absence:presence of tumor[ Although MEL!water dose is pharmacological "Perissin et al[\ 0882#\ it is not toxic "Maestroni + Conti\ 0889^ Mocchegiani et al[\ 0885#\ but may mimic physiological control mechanisms of the pineal gland because of MEL|s short half!life "Lang\ Aubert\ Conne\ Bradtke + Sizonenko\ 0872#[ In this context\ three important questions arise] _rstly the presence of circulating MEL in hybrid BD1F0 mice because of no circulating MEL in C46BL:5 mice "Ebihara et al[\ 0875#^ secondly the e}ective amount of MEL!water intake during the dark period either because the half!life of MEL is short "01Ð39 min[# "Lang et al[\ 0872#\ or because mice drink normal tap water during the light period[ Third the real e}ects on metastasis volume\ corticosterone\ zinc and immune parameters due to surgical stress "pinealectomy# and:or RS "Prasad\ 0874^ Giraldi et al[\ 0883a\b#[ The observed e}ects are only related to RS because of no di}erences in corticosterone after 1 months from pinealectomy between shamÐpx and intact mice and between px and px stressed mice[ Circulating MEL has been clearly demonstrated in young inbred C46BL:5 mice "Conti + Maestroni\ 0885#\ including young!adult hybrid BD1F0 mice "Perissin et al[\ 0882\ Giraidi et al[\ 0883a\b# used here[ Indeed undetectable night urinary MEL secretion is observed in young hybrid BD1F0 mice even after functional pinealectomy "³19 vs 096 pg:mouse:01 h in young control# "Perissin et al[\ 0882#[ Finally\ total MEL!water uptake during the dark period is 3[7Ð4 ml:night:mouse\ corresponding to MEL!water amount uptaken by old mice "4 ml:night:mouse# when normal tap water is deprived during the light period "Pierpaoli + Regelson\ 0883^ Mocchegiani et al[\ 0883a\ 0887#[ Thus\ MEL! water uptake occurs in hybrid BD1F0 mice independently by a possible major consumption of water during the light period and despite the bitter taste of MEL "Mocchegiani et al[\ 0887#[ MEL treatment was not performed on shamÐpx tumor bearing mice because of no di}erences in volume metastasis\ and in immunological and nutritional parameters with intact tumor bearing mice\ as previously shown between intact and shamÐpx¦MEL mice "Mocchegiani et al[\ 0885#[ No in~u! ence of dietary intake because body weights are similar in treated and control mice "data not shown#[ Metastasis volume reduction only occurs in stressed intact and px tumor bearing mice[ The restoration of metabolic zinc pool and thymic functions by MEL\ via corticosterone\ may be\ thereby\ more involved for three reasons[ "0# High corticosterone levels and the reduced metabolic zinc pool in old and px mice are restored by MEL treatment "Mocchegiani et al[\ 0883a\ 0885#\ "1# MEL nocturnal peak restoration in old\ MEL treated mice is strictly related with normalization of nocturnal peaks of zinc and ZnFTS\ via corticosterone "Mocchegiani et al[\ 0887#\ and "2# In vitro experiments from old thymic explants show a direct action of zinc\ rather than MEL\ on restoring thymic functions "Mocchegiani et al[\ 0887#[ The presence of signi_cant correlations "negative or positive# between zinc and corticosterone or ZnFTS exclusively in stressed intact and px tumor bearing mice after MEL treatment\ lends further support to this interpretation[ MEL counteracts a!1 adrenergic immunosuppression on peripheral blood lymphocytes "Lieb! mann\ Hofer\ Felsner\ Wolfer + Schauenstein\ 0885#[ Adrenergic system is controlled by glu! cocorticoids in stress "Mobley\ Manier + Sulser\ 0872# and involved on cytokine production by activated lymphocytes "Besedovsky + Del Rey\ 0881#[ These _ndings\ together with the interplay between zinc and MEL\ via glucocorticoids\ "Mocchegiani et al[\ 0883a\ 0885\ 0887#\ which is\ in 31 E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35 turn\ involved in adrenergic receptors modulation "Viticchi\ Bulian\ Pierpaoli + Piantanelli\ 0883^ McLeod + Cairncross\ 0884#\ and the role of zinc\ MEL and glucocorticoids for IL!1 activity "Tanaka et al[\ 0889^ Caroleo et al[\ 0881^ Hadden\ 0884^ Mocchegiani et al[\ 0885#\ suggest the presence of an immunoregulatory circuit where MEL\ via glucocorticoids and the adrenergic system\ may be relevant for major zinc bioavailability in MEL stressed\ tumor bearing mice[ As such\ increments of IL!1 from activated lymphocytes may occur[ In vitro^ studies showing zinc to increase IL!1 production from activated lymphocytes of cancer patients "Prasad et al[\ 0886a#\ con_rm this assumption[ Thus IL!1 increments due to major zinc bioavailability in tumor stressed mice is not an epiphenomenon but a speci_c signal against tumors\ as suggested in human cancer "Prasad et al[\ 0886a\b#[ This con_rms the relevance also of circulating IL!1 as a good immunological marker in zinc!de_ciency cancer condition\ as previously suggested "Prasad et al[\ 0886a#[ The presence of signi_cant positive or inverse correlations between zinc or corticosterone and IL!1\ respectively\ in intact and px stressed mice showing metastasis reduction\ support this interpretation[ Because mRNA level of IL!1 is decreased when Jurkat cells "malignant T!lymphoid cells# grow in zinc!de_cient medium "Prasad\ 0882#\ is a further indirect evidence of that[ Thus\ an action of MEL as antistress agent may be supported in order to induce major zinc biovailability for thymic e.ciency and IL!1 production\ which is\ in turn\ involved in zinc!uptake by thymulin! epithelial cells and consequently in the production of ZnFTS molecules "Saha\ Hadden + Hadden 0884\ Hadden\ 0884#[ The major zinc bioavailability may occur by the suppressive e}ect of MEL on glucocorticoid pathway "Gupta\ 0889#\ which\ in turn\ induces a zinc!depleting e}ect "Prasad\ 0874^ Fabris\ Mocchegiani + Provinciali\ 0886#[ Alternatively\ the presence of MEL receptors in gut cells may be involved "Lee + Pang\ 0880#[ Zinc!_nger proteins a}ect IL!1 gene expression in T lymphocytes "both circulating and into the tumor# "Owaki et al[\ 0882^ Skerka\ Decker + Zipfel\ 0884^ Nagashima et al[\ 0886#[ This _nding with our data are suggestive to assign peculiar role to zinc for IL!1 production\ which\ in turn\ may reactivate anergic natural killer cells against tumor "Agrawal\ Kranz\ Reddish + Longenecker\ 0887#[ Zinc induces also apoptosis of tumor cells "Provinciali\ Di Stefano\ Stronati + Fabris\ 0885a#\ whereas MEL is less e}ective both in vivo and in vitro models "Sainz et al[\ 0884^ Provinciali\ Di Stefano\ Bulian\ Tibaldi + Fabris\ 0885b#[ Possible MEL apoptotic role against tumor may occur by means of a decrement of NF!kB DNA binding activity "Chuang\ Mohan\ Metz + Reiter\ 0885#\ which is\ in turn\ under the control of TNF!a "Wang\ Mayo + Baldwin\ 0885#[ Because TNF!a is reduced in MEL treated mice "Ben! Nathan\ Maestroni + Conti\ 0886#\ the apoptotic role of MEL is still unclear[ Thus\ without discrediting MEL|s direct oncostatic role\ MEL is more an immunomodulating agent\ via zinc\ against tumors\ this warrants further work[ In conclusion\ the interplay between zinc and MEL\ via corticosterone\ for normal immune response\ including IL!1\ already proposed during devel! opment and aging "Mocchegiani et al[\ 0883a\ 0885\ 0887#\ may be also suggested in cancer with this possible physiological cascade] MEL treatment : reduction of corticosterone release : major zinc bioavailability : consequent normal immune response[ Because of no di}erences in increased survival between old zinc and old MEL treated mice "Mocchegiani et al[\ 0887#\ zinc treatment with chemotherapy in cancer patients\ other than MEL "Lissoni et al[\ 0884#\ may be taken into account[ MEL is an antioxidant "Reiter\ 0883# with a protective role on stem cells against toxicity by antiblastic drugs "Maestroni et al[\ 0883#[ Because also zinc is an antioxidant "Willson\ 0866#\ with a protective role against chemotherapic toxicity by means of zinc!bound met! allothioneins "Lazo et al[\ 0880#\ the proposal of physiological zinc treatment with chemotherapy E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35 32 in cancer "Hadden\ 0884^ Fabris + Mocchegiani\ 0884\ Prasad et al[\ 0886a\b# might _nd further rationale[
Aknowledgments
This work was supported by the Italian National Research Council "CNR# Special Project {ACRO| No[ 85[99463[PF28\ by MURST 39 and 59) to Professor Tuilio Giraldi\ U[S[U[ 0884\ by I[N[R[C[A[ and by Health Italian Ministry[ The generosity of Fondazione Calliero\ Trieste\ Italy\ in providing the protected animal housing facility is gratefully appreciated[ The authors thank Mrs N[ Gasparini\ Mr F[ Marchegiani\ Mr G[ Bernardini and Mr G[ Mazzarini for their excellent technical assistance[
References
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LAK cells and on the sensitisation of tumor cells to the killing mediated by LAK cells[ European Cytokine Network\ 6\ 433Ð434[ Provinciali\ M[\ Di Stefano\ G[\ Bulian\ D[\ Tibaldi\ A[\ + Fabris\ N[ "0885b#[ E}ect of melatonin and pineal grafting on thymocytes apoptosis in aging mice[ Mech[ A`in` Develop[\ 89\ 0Ð08[ Reiter\ R[ J[\ Tan\ D[ X[\ Poeggeler\ B[\ Chen\ L[ D[\ + Menendez!Pelaez\ A[ "0883#[ Melatonin\ free radicals and cancer initiation[ In G[ J[ M[ Maestroni\ A[ Conti + R[ J[ Reiter "Eds[#\ Advances in Pineal Research Vol[ 6\ "pp[ 100Ð117#[ London\ U[K[] John Libbey and Company Ltd[ Riley\ V[\ Fitzmaurice\ M[ A[\ + Spackman\ J[ "0870#[ Psychoneuroimmunologic factors in neoplasia] studies in animals[ In R[ Ader "Ed[#\ Psychoneuroimmunolo`y "pp[ 20Ð091#[ New York\ N[Y[] Academic Press[ Sa_eh\ B[\ Kendall\ M[ D[\ Norman\ J[ C[\ Metreau\ E[\ Dardenne\ M[\ Bach\ J[ F[\ + Pleau\ J[ M[ "0889#[ A new radioimmunoassay for the thymic peptide thymulin\ and its application for measuring thymulin in blood samples[ J[ Immunol[ Methods\ 016\ 144Ð151[ Saha\ A[ R[\ Hadden\ E[ M[\ + Hadden\ J[ W[ "0884#[ Zinc induces thymulin secretion from human thymic epithelial cells in vitro and augments splenocytes and thymocytes response in vivo[ Int[ J[ Immunopharmacol[\ 06\ 618Ð622[ Sainz\ A[ P[\ Mayo\ J[ C[\ Uria\ K[\ Katler\ M[\ Antolin\ J[\ Rodriguez\ C[\ + Menendez!Pelaez\ A[ "0884#[ The pineal hormone melatonin prevents in vivo and in vitro apoptosis in thymocytes[ J[ Pineal Res[\ 08\ 067Ð077[ Shellard\ S[ A[\ Whelan\ R[ D[ H[\ + Hill\ B[ T[ "0878#[ Growth inibitory and cytotoxic e}ects of melatonin and its metabolises on human tumor cell lines in vitro[ Brit[ J[ Cancer\ 59\ 177Ð189[ Skerka\ C[\ Decker^ E[ L[\ + Zipfel\ P[ F[ "0884#[ A regulatory element in the human interleukin!1 gene promoter is a binding site for the zinc _nger proteins SP0 and EGR!0[ J[ Biol[ Chem[\ 169\ 11499Ð11495[ Skwarlosonta\ K[ "0885#[ Functional connections between the pineal gland and immune system[ Acta Neurobiol[ Exp[\ 45\ 230Ð246[ Slominski\ A[\ + Pruski\ D[ "0882#[ Melatonin inhibits proliferation and melanogenesis in rodent melanoma cells[ Exp[ Cell Res[\ 195\ 078Ð083[ Stanberry\ L[ R[\ Das Gupta\ T[ K[\ + Beattie\ C[ W[ "0872#[ Photoperiodic control of melanoma growth in Hamsters] in~uence of pinealectomy and melatonin[ Endocrinolo`y\ 002\ 358Ð364[ Stankov\ B[\ Lucini\ V[\ Scaglione\ F[\ Cozzi\ B[\ Righi\ M[\ Canti\ G[\ Demartini\ G[\ + Fraschini\ F[ "0880#[ 1! ð014IŁ Iodomelatonin binding in normal and neoplastic tissues[ In F[ Fraschini + R[ J[ Reiter "Eds[#\ Role of melatonin and pineal peptides in neuroimmunomodulation "pp[ 006Ð014#[ New York\ N[Y[] Plenum Press[ Tanaka\ Y[\ Shiozawa\ S[\ Moromoto\ I[\ + Fujita\ T[ "0889#[ Role of zinc in interleukinÐ1 "IL!1#!mediated T!cell activation[ Scand[ J[ Immunol[\ 20\ 436Ð441[ Viticchi\ C[\ Bulian\ D[\ Pierpaoli\ W[\ + Piantanelli\ L[ "0883#[ Brain cortex a! and b!adrenoceptors are di}erentially modulated by pineal graft in aging mice[ Ann[ N[Y[ Acad[ Sci[\ 608\ 337Ð342[ Vogel\ V[ H[\ + Bower\ D[ B[ "0880#[ Stress\ immunity and cancer[ In N[ Plotniko}\ A[ Murgo\ R[ Faith + J[ Wlbron "Eds[#\ Stress and Immunity "pp[ 382Ð496#[ Boca Raton\ Florida\ U[S[A[] CRC Press[ Wang\ C[ Y[\ Mayo\ M[ W[\ + Baldwin\ A[ S[ Jr[ "0885#[ TNF and cancer therapy!induced apoptosis potentiation by inhibition of NF!kB[ Science\ 163\ 673Ð676[ Willson\ R[ L[ "0866#[ Zinc] a radical approach to disease[ New Scientist\ 2\ 447Ð459[